Ligation-Recutting Efficiency

To determine the functional purity of an enzyme, a ligation-recutting assay was conducted. As shown in the chart below, the high quality of Takara's restriction enzymes are guaranteed by the criteria.

Contamination of ligase inhibitor, phosphatase and exonuclease was detected by the following procedures:

At first, overdigestion was performed by applying 2-fold to 50-fold excessive amount of restriction enzymes to a substrate DNA. Digested DNA was recovered and diluted to T4 DNA Ligase Buffer [66 mM Tris-HCl (pH7.6), 6.6 mM MgCl₂, 10 mM DTT, 0.4 mM ATP] at a 5'-termini concentration of 0.1-1.0 µM. After an adequate amount of T4 DNA ligase was added, it was incubated at 16°C for 1 hour or 16-18 hours. The recovered DNA was diluted to a reaction buffer again and recut by the same restriction enzyme.

*Digested DNA was ligated by TaKaRa DNA Ligation Kit, Ver.2 and incubated at 26°C for 10 min.